Cultures of E1-immortalized cells and processes for culturing the same to increase product yields therefrom

ABSTRACT

The invention provides processes for culturing cells derived from embryonic retinoblast cells immortalized by adenovirus E1 sequences, preferably PER.C6® cells, to improve product yields from such cells. Feed strategies for such cells and cultures with very high cell densities are provided, resulting in high yields of products, such as recombinant antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 11/259,245filed Oct. 26, 2005, pending, which is a continuation of InternationalPatent Application PCT/EP2004/050724, filed May 6, 2004, designating theUnited States of America, published, in English, as International PatentPublication WO 2004/099396 A1 on Nov. 18, 2004, which itself claimspriority from International Patent Application Nos. PCT/EP03/50155 filedMay 9, 2003, PCT/EP03/50390 filed Sep. 1, 2003, PCT/EP03/50940 filedDec. 4, 2003, and PCT/EP04/050061 filed Jan. 30, 2004, the contents ofthe entirety of each of which are incorporated herein by this reference.

FIELD OF THE INVENTION

The invention relates generally to biotechnology in area of cellculture. In particular, the invention relates to the field of culturingcells derived from cells that have been immortalized with E1 sequencesfrom adenovirus. More in particular, the invention relates to culturingsuch cells to obtain high levels of products from such cells.

BACKGROUND OF THE INVENTION

A human PER.C6® cell line, exemplified by cells deposited at the ECACCunder No. 96022940, derived from retina cells by immortalization withthe adenovirus (Ad5) E1a and E1b genes is disclosed in U.S. Pat. No.5,994,128. Besides the ability to function as packaging cells forE1-deleted adenoviral vectors (U.S. Pat. No. 5,994,128; WO 01/005945),and for producing other viruses (WO 01/38362), E1-immortalized cells,such as PER.C6 cells, can be used to produce recombinant proteins, suchas antibodies (WO 00/63403).

Xie et al. (2002) have disclosed a process for serum-free suspensioncultivation of E1-immortalized cells. However, the product yieldsobtained using the culturing processes disclosed in the art forE1-immortalized cells, can be improved.

SUMMARY OF THE INVENTION

In particular embodiments, the present invention provides processes toincrease the product yield from E1-immortalized cells.

In certain embodiments, the invention provides feed strategies forfed-batch or fed-perfusion cultures of cells immortalized by adenovirusE1 sequences. In one embodiment thereof, the invention provides a methodfor the culturing of such cells, the cells capable of growing insuspension, comprising the steps of: determining at least once duringthe culturing of the cells the concentration of at least one mediumcomponent selected from the group consisting of glucose, glutaminephosphate, leucine, serine, isoleucine, arginine, methionine, cystine,valine, lysine, threonine and glycine, adding components to the mediumduring the culturing of the cells at or prior to the depletion of atleast one of the components of which the concentration was determined inthe previous step, wherein the components added at least compriseglucose, glutamine, phosphate, leucine, serine, isoleucine, arginine,methionine, and cystine. Other components that beneficially may be addedaccording to the invention, amounts and time of addition of thecomponents are provided herein below, as well as in the claims.

In another embodiment, the invention provides a culture of cells derivedfrom cells immortalized by adenovirus E1 sequences, characterized inthat the culture comprises at least 10×10⁶ cells/ml. Preferably, theculture comprises at least 12×10⁶ cells/ml, more preferably, at least15×10⁶ cells/ml. In certain preferred embodiments, the culture accordingto the invention comprises more than 20×10⁶, 25×10⁶, 30×10⁶ or 40×10⁶cells/ml. Methods to obtain such cultures are also provided herein.

In yet another aspect, a method to increase cell densities and productyields from a culture of cells immortalized by adenovirus E1 sequencesis provided. In one embodiment hereof, a process for culturing suchcells is provided, characterized in that the process comprises a step ofsubculturing the cells at a seeding concentration of between 0.8×10⁶ and2.0×10⁶ viable cells/ml, preferably, between 0.9×10⁶ and 1.5×10⁶ viablecells/ml.

Preferably, the cells used in the methods of the invention are derivedfrom retina cells, more preferably, from human embryonic retina (HER)cells, such as the cells deposited under ECACC No. 96022940. In certainembodiments, the cells are PER.C6 cells.

In certain embodiments, the cells can produce recombinant proteins,preferably, antibodies, at high yields. In other embodiments, the cellscomprise recombinant adenoviral vectors having a deletion in theE1-region, or other viruses, which can be produced on the cells in highyields using the process according to the invention. In preferredembodiments, the cells are cultured at least part of the time in aserum-free medium.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Graph showing growth and antibody production of a PER.C6 clone(clone 1) grown in shake flask at two different starting cellconcentrations (0.3 and 1.0×10⁶/ml). Left vertical axis: viable cellnumber (N_(v)). Right vertical axis: antibody (Ab) concentration.Horizontal axis: time (hours).

FIG. 2. Graph showing the decrease in the cell specific utilization ofglutamine with increasing viable cell numbers for an antibody-producingPER.C6® clone (clone 1). N: cell number.

FIG. 3. Profiles for a batch culture of PER.C6® clone 1. A: Metabolites.Glc, glucose; Lac, lactate; Gln, glutamine; NH₃, ammonia; P, phosphate.B-E: Amino acids (AA).

FIG. 4. Graph showing the effect of a feed component mix containingglucose, glutamine, amino acids, phosphate, calcium and growth factorson PER.C6 clone 1. A: viable cell numbers (N_(V)). B: antibody (Ab)concentration. Circles: batch. Squares: fed-batch.

FIG. 5. Graph showing viable cell numbers (N_(V)) and antibody (Ab)yields of PER.C6 clones where culture medium was completely exchangedonce per day (two independent experiments each). A: clone 1. B: clone 2.

FIG. 6. Result of modified feed (example 4) for clone 1. A: viable cellnumbers (N_(V)). B: antibody (Ab) concentration. Open circles: batch.Closed circles: fed-batch.

FIG. 7. Result of further improved modified feed with different firstand subsequent feed additions (Example 4, Table 2) for clone 1. A:viable cell numbers (N_(V)). B: antibody (Ab) concentration. Circles:batch. Squares: fed-batch. Arrows: last feed.

FIG. 8. Result of further improved modified feed with different firstand subsequent feed additions (Example 4, Table 2) for clone 2. N_(V):viable cell number. Ab: antibody concentration.

FIG. 9. Galactosylation levels of IgG produced according to processesaccording to invention.

FIG. 10. Result of further improved modified feed with different firstand subsequent feed additions (Example 4, Table 2) for clone 3. N_(V):viable cell numbers. Ab: antibody concentration.

DETAILED DESCRIPTION OF THE INVENTION

The productivity of any cell line is mainly defined by three basicparameters, the specific productivity of the cell line, the peak viablecell concentration that is attainable and the length of the productionprocess that is possible. Increases in either of these variables willlead to increases in the final product concentration and is dependent toa large extent on the cell line. In a straight batch culture, cell linessuch as CHO and SP2/0 can achieve cell densities up to 4×10⁶/ml. Infed-batch or perfusion processes the viable cell concentration isincreased, and typically hybridoma cells such as SP2/0 can be culturedup to 10×10⁶ cells/ml, while CHO can be cultured up to 6-10×10⁶cells/ml. The invention describes methods to increase the viable celldensity of cultures of cells immortalized by adenovirus E1 sequences,preferably, derived from embryonic retina cells, to attain celldensities beyond those reported in the prior art. Furthermore, themethods according to the invention can be used to obtain higher productyields from cultures of cells according to the invention.

Disclosed herein are improvements in how E1-immortalized cells, such asPER.C6® cells (available from Crucell, Leiden, NL), can advantageouslybe used for the production of high yields of monoclonal antibodies. Itis disclosed that these cells be cultured to very high viable cellconcentrations in a straight batch process (e.g., up to 14×10⁶ viablecells/ml).

Furthermore, E1-immortalized cells, such as PER.C6® cells, are wellsuited to a fed-batch process as a culture of these cells unexpectedlyconsumes lactate and ammonia and maintains viability for long periods oftime under nutrient limiting conditions. Methods to increase productyields from the cells by a feed strategy in cultures are providedherein.

With the term “feed strategy” as used herein is meant the addition ofcertain identified components including, but not limited to, nutrients,such as sugars, amino acids, and the like, to the culture medium. Theidentified components are, preferably, added in certain amounts and atcertain times, when they are required to improve product yields from thecells, such as provided herein.

E1-immortalized cells, such as PER.C6® cells, are also well suited to aperfusion process as they can be maintained at very high viable cellconcentrations (up to 50×10⁶ cells/ml with a viability of at least 85%)for long periods of time and with good final product concentrations.

Culture Media

The processes of the invention generally increase the product yieldsfrom the cells compared to yields obtained with processes described inthe art for the cells according to the invention. Preferably, serum-freeculture media are used at least part of the time in the processesaccording to the invention. Preferably, the medium contains onlyrecombinantly produced proteins, which are not of animal origin. Suchculture media are commercially available from various sources. In oneembodiment of the invention, VPRO culture medium (JRH Biosciences) isused for the fed-batch or (fed-)perfusion process.

Products

The methods of the invention are, preferably, used to produce productsin cells of the invention. The processes of the present invention can beused for the improved production of antibodies, as well as otherproteins (WO 00/63403, the contents of which are incorporated herein bythis reference). For the production of proteins, the cells of theinvention suitably comprise nucleic acid encoding the proteins, inoperable association with elements capable of driving expression of theproteins. Furthermore, the processes can be used for improvement of theproduction of recombinant adenoviral vectors having a deletion in theE1-region, in which case the cells are used as complementing cells,which in itself is known to the skilled person according to establishedmethodology (e.g., U.S. Pat. No. 5,994,128; WO 01/005945, the contentsof both of which are incorporated herein by this reference). Moreover,the processes according to the invention can be used to improve aprocess for propagation of other (non-adenovirus) viruses in the cells(WO 01/38362, the contents of which are incorporated herein by thisreference). Hence, products according to the invention can berecombinant proteins, such as antibodies, erythropoietin, and the like,as well as recombinant adenoviral vectors with a deletion in the E1region, or other viruses.

Cells

The cells according to the invention are cells that have beenimmortalized with E1 sequences from an adenovirus, which cells are alsoreferred to herein as E1-immortalized cells. Such cells express at leasta functional part of the E1A region of an adenovirus, and, preferably,also at least a functional part of the E1B region. E1A protein hastransforming activity, while E1B protein has anti-apoptotic activities.The cells according to the invention may be derived from any cell,including lung cells, kidney cells, amniocytes, but, preferably, arederived from retina cells. They may be derived from embryonic retinacells. Preferably, the cells according to the invention are human cells.A method for immortalization of embryonic retina cells has beendescribed in the art (U.S. Pat. No. 5,994,128). Accordingly, a retinacell that has been immortalized with E1 sequences from adenovirus can beobtained by that method. In certain preferred embodiments, the cells ofthe invention are derived from E1-immortalized HER cells, such as PER.C6cells. PER.C6 cells for the purpose of the present application shallmean cells from an upstream or downstream passage or a descendent of anupstream or downstream passage of cells as deposited under ECACC No.96022940. In addition, also the E2A region with a ts125 mutation may bepresent (see e.g., U.S. Pat. No. 6,395,519, the contents of which areincorporated herein by this reference) in the cell. A cell derived froma PER.C6 cell can be a PER.C6 cell infected with recombinant adenovirusor other virus, and can also be a PER.C6 cell into which recombinantnucleic acid has been introduced, for instance, comprising an expressioncassette wherein nucleic acid encoding a protein of interest is operablylinked to sequences capable of driving expression thereof, such as apromoter and polyA signal, wherein, preferably, the cells are from astable clone that can be selected according to standard procedures knownto the person skilled in the art. A culture of such a clone is capableof producing a protein encoded by the recombinant nucleic acid.

Components for Feed Strategies

In one aspect, the invention provides processes for culturing cellsaccording to the invention, wherein by feed strategies according to theinvention certain amino acids are added during the culturing process toreplenish amino acids of which the concentration has become or willbecome limiting for an optimal process and product yields. By amino acidis intended all naturally occurring alpha amino acids in both their Dand L stereoisomeric forms, and their derivatives. A derivative isdefined as an amino acid that has another molecule or atom attached toit. Derivatives would include, for example, acetylation of an aminogroup, amination of a carboxyl group, or oxidation of the sulfurresidues of two cysteines to form cystine. Further, amino acidderivatives may include esters, salts, such as chlorides, sulphates, andthe like, as well as hydrates. It will be understood by the personskilled in the art, that where a specific amino acid is mentionedherein, a derivative may also be used and is meant to be included withinthe scope of the invention. Other components such as sugars, growthfactors, vitamins, etc., may also be added to improve the processesaccording to the invention.

Feed Strategies

In one aspect, the invention provides a method for producing a productin cells immortalized by adenovirus E1 sequences, in a culture medium,wherein the product is chosen from the group consisting of a recombinantprotein, a virus, and a recombinant adenovirus with a deletion in the E1region, characterized in that the method comprises a step wherein atleast leucine, serine, isoleucine, arginine, methionine and cystine areadded to the culture medium. In one aspect the invention provides amethod for the culturing of cells immortalized by adenovirus E1sequences, the cells capable of growing in suspension, comprising thesteps of: determining at least once during the culturing of the cellsthe concentration of at least one medium component selected from thegroup consisting of glucose, glutamine, phosphate, leucine, serine,isoleucine, arginine, methionine, cystine, valine, lysine, threonine andglycine, adding components to the medium during the culturing of thecells at or prior to the depletion of at least one of the components ofwhich the concentration was determined in the previous step, wherein thecomponents added at least comprise glucose, glutamine, phosphate,leucine, serine, isoleucine, arginine, methionine and cystine.“Depletion” as used herein is defined as the time a component has aconcentration of 30% or less of the starting concentration in theculture medium. In these aspects, the determination of the concentrationof at least one medium component selected from the group consisting ofglucose, glutamine, phosphate, leucine, serine, isoleucine, arginine,methionine or cystine is preferred over the determination of onlycomponents selected from the group consisting of valine, lysine,threonine and glycine. In certain embodiments, the concentration of atleast two medium components according to the invention is determined inthe first step. In certain embodiments, the components that are addedfurther comprise one or more of valine, lysine, threonine, glycine,asparagine, tyrosine, histidine, phenylalanine, tryptophan, calcium,LongR3 IGF-1, Long EGF and insulin. In specific embodiments, thecomponents are added in an end concentration in mmoles/l of freshlyadded component per 10×10⁶ cells/ml of 6.0 for glucose, 2.60 in thefirst feed and 1.75 in subsequent feeds for glutamine, 0.70 forphosphate, 0.66 for leucine, 1.10 in the first feed and 0.55 insubsequent feeds for serine, 0.50 for isoleucine, 0.46 for arginine,0.23 for methionine, and 0.25 for cystine. In further embodiments, thefollowing components are further added to an end concentration inmmoles/l of freshly added component per 10×10⁶ cells/ml of 0.45 forvaline, 0.44 for lysine, and 0.30 for threonine. In further embodiments,the following components are further added to an end concentration inmmoles/l of freshly added component per 10×10⁶ cells/ml of 0.10 forasparagine, 0.13 for tyrosine, 0.10 for histidine, 0.02 forphenylalanine, and 0.06 for tryptophan. Furthermore, calcium may beadded in an end concentration in mmoles/l of freshly added component per10×10⁶ cells/ml of 0.02. Growth factors such as IGF, EGF, and insulin ortheir derivatives may also suitably be present in the growth medium. Theamounts for the addition of components above may have an error marginper component of 33% or less, preferably, 20% or less, more preferably,10% or less, even more preferably, 5% or less. The amounts are presentedper 10×10⁶ cells/ml, and are linearly dependent on the number ofcells/ml. In preferred embodiments, the components are added at between48 hours and the moment of depletion of at least one of the mediumcomponents the concentration of which was determined in the previousstep. In certain embodiments, the addition is at a time between 24 hoursand just prior to depletion. In certain aspects, the invention providesa method according to the invention, wherein the cells express arecombinant immunoglobulin that is secreted into the culture medium to alevel of at least 500 mg per liter, preferably, at least 700 mg/l, morepreferably, at least 850 mg/l, even more preferably, at least 1000 mg/l,still more preferably, at least 1250 mg/l, still more preferably, atleast 1500 mg/l, still more preferably, at least 1750 mg/l and stillmore preferably, at least 2000 mg/l. In general, the addition of mediumcomponents according to the invention in, for instance, a fed-batchprocess, results in an increase in the yield of produced product of atleast 1.5×, preferably, at least 2×, more preferably, at least 2.5× andstill more preferably, about 3× or even higher, compared to the processwherein no components are added, i.e., the batch process.

In addition to use in a fed-batch process, the feed strategies of theinvention can also be beneficially used in an optimized batch process,as set out in Example 5.

Perfusion

Alternatively, in another aspect of the invention, the entire culturemedium may be exchanged. It is shown that unexpected high viable celldensities can be attained when this is applied to cells derived fromretina cells immortalized by adenovirus E1 sequences. Exchanging culturemedium may be performed by any means known to the person skilled in theart, including, but not limited to, collection of the cells bycentrifugation, filtration, and the like, followed by re-suspension ofthe cells into fresh culture medium. Alternatively, a perfusion systemmay be used, wherein culture medium is either continuously orintermittently exchanged using a cell separation device such as aCentritech centrifuge or passage through a hollow fiber cartridge, andthe like. It is, therefore, another aspect of the invention to provide aprocess for culturing cells derived from embryonic retina cellsimmortalized by adenovirus E1 sequences, characterized in that culturemedium is exchanged at a rate of 0.2-3, preferably, 0.5-3, culturevolumes per day (24 hours). Cultures obtained using this method,preferably, have viable cell densities higher than 20×10⁶ cells/ml, morepreferably, higher than 30×10⁶ cells/ml. In certain aspects, suchcultures have cell densities higher than 40×10⁶ cells/ml. In certainaspects such cultures are used to produce recombinant antibodies with ayield of at least 150 mg/l/day, preferably, at least 200 mg/l/day, morepreferably, at least 300, 400, or 500 mg/l/day. Of course, also otherproducts according to the invention can be produced by such methods. Itis shown here that one complete volume exchange of culture medium eachday supports at least 30×10⁶ viable cells/ml with antibody yields ofmore than 500 mg/L/day (up to 750 mg/l/day) (FIG. 5). One completemedium exchange per day corresponds to a continuous perfusion rate ofthree volumes per day, meaning that a continuous perfusion system couldyield approximately at least 150-200 mg/L/day. One method to reduce thisperfusion rate and, thus, increase antibody yields (by reducing thevolume in which the antibody is secreted) is to supplement the freshculture medium with the essential components (known as fed-perfusion).These components for antibody-producing E1-immortalized cell, such asPER.C6 cell, clones are identified herein (see Example 2) and thereforeit is another aspect of the present invention to provide such afed-perfusion system, wherein the feed strategies, according to thepresent invention, are employed. A common drawback of fed-perfusionprocesses is the build-up of toxic metabolic by-products (such aslactate and ammonia), which can result in low cell viabilities andproduct yields. There is often a requirement at high cell concentrationsfor a high perfusion rate to remove these by-products. One advantagedemonstrated for E1-immortalized cell, such as PER.C6 cell, clonesaccording to the invention is that they are capable of utilizing lactateand ammonia such that concentrations do not become problematical (see,FIG. 3A). It is, therefore, possible to obtain an antibody yield of atleast 500 mg/l/day by changing the culture medium once or twice a day.Alternatively, this can be achieved by using a continuous perfusion rateof, for instance, one volume per day in combination with supplementationof the medium with a feed concentrate (fed-perfusion). This canadvantageously be combined with a cell bleed (removing a certainpercentage of the cells population).

Cultures with high cell densities are advantageous for obtaining highproduct yields. It is, therefore, another aspect of the invention toprovide a culture of cells derived from cells immortalized by adenovirusE1 sequences, the culture comprising at least 10×10⁶ cells/ml. Theviability in the culture is at least 80%. Preferably, the viability isat least 90%, more preferably, at least 95%. The cultures according tothe invention are, preferably, suspension cultures, meaning that thecells in the cultures are in suspension in the culture medium, such asin shake flasks, roller bottles, bioreactors, including stirred tanks,air lift reactors, and the like. The strategies disclosed herein may,however, also be used for cultures of cells in hollow fiber reactors,such as described by Tanase et al. (1997), and for adherent cultures,such as cells on microcarriers. In one embodiment, the culture comprisesat least 12×10⁶ cells/ml. It is disclosed herein that up to 14×10⁶cells/ml can be obtained by a straight batch culture.

It is further demonstrated that, using medium perfusion, even highercell densities can be achieved, for example, up to 50×10⁶ cells/ml. Theprior art does not provide any indication that such unexpected high celldensities are obtainable. In other preferred embodiments, therefore, theinvention provides a culture of cells derived from E1-immortalizedcells, preferably, derived from retina cells, the culture comprising atleast 15×10⁶ cells/ml, preferably, at least 20×10⁶ cells/ml, morepreferably, at least 25×10⁶ cells/ml. In specific embodiments, theculture comprises at least 30×10⁶ cells/ml, or even at least 40×10⁶cells/ml. Cultures with at least 15×10⁶ cells/ml, according to theinvention, appear obtainable by a perfusion process, meaning thatculture medium is exchanged during the culturing process. The cultures,according to the invention, have a viability of at least 80%,preferably, at least 85%, more preferably, at least 90%, still morepreferably, at least 95%. The cultures are suspension cultures. Thecultures further comprise growth medium. The growth medium, preferably,is serum-free. The cells of the culture may comprise recombinant nucleicacid molecules encoding immunoglobulins, or parts or derivativesthereof, in expressible format. Such cells are capable of producingimmunoglobulins in high yields. In particular, it is shown herein that aculture of cells, according to the invention, wherein the medium isexchanged every day, and wherein more than 30×10⁶ cells/ml are present,can provide recombinant antibody yields of at least 500 mg/l/day. Thecells in the culture, preferably, produce at least 10 pgprotein/cell/day.

The processes of the invention, especially those for recombinant proteinproduction, can also be combined with other measures described in theart that in some cases improve product yields. Therefore, in certainembodiments of the invention, the culture medium is subjected to atemperature shift before or during the production phase, e.g., byrunning the process at a lower temperature, for instance, between 30° C.and 35° C., in the production phase (see e.g., U.S. Pat. No. 6,506,598,the contents of which are incorporated herein by this reference, andliterature cited therein, which describes effects of lowering the cellculture temperature on several parameters for recombinant proteinproduction), or by the addition of cold culture medium to the culture(wherein cold is meant to be lower than the temperature the cells arecultured in, preferably, the cold culture medium having a temperaturebetween 2° C. and 8° C.) when the cells are subcultured or later duringthe culture process. In other embodiments, specific growth factors maybe added to improve the processes according to the invention with regardto product yields. In yet other embodiments, for the production ofproteins, the processes, according to the invention, may be improved bythe addition of alkanoic acids or salts thereof, such as sodiumbutyrate, either during the whole culture phase or only during theproduction phase (see, e.g., U.S. Pat. No. 6,413,746, and referencestherein, which describes effects of addition of butyrate on productionof proteins in cell culture). In yet other embodiments for theproduction of proteins, the culture medium is subjected to a temperatureor pH shift (Weidemann et al., 1994, Sauer et al., 2000).

It will be clear to the person skilled in the art that several aspectsand/or embodiments, according to the invention, can be combined toprovide a process for culturing cells which leads to particularly goodproduct yields. As a non-limiting example, it is, for instance, possibleto seed a culture of E1-immortalized cells at about 0.8×10⁶ to 2.0×10⁶cells/ml, and use a feed strategy and/or exchange the growth mediumduring the culturing process to improve the final product yields.

The invention will now be illustrated with some examples, not intendedto limit the scope of the invention.

Experimental

Methods and vectors for genetically engineering cells and/or cell linesto express a protein of interest are well known to those of skill in theart; for example, various techniques are illustrated in CurrentProtocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, NewYork, 1988, and quarterly updates) and Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989).General and standard cell culture techniques are known to the personskilled in the art, and are, for instance, described in R. I. Freshney,Culture of animal cells: A manual of basic technique, fourth edition(Wiley-Liss Inc., 2000, ISBN 0-471-34889-9). Such standard techniqueswere followed unless otherwise noted.

Cell Culture Protocols

PER.C6® cells were cultured in the examples. Cells were adapted fromadherent cultures in DMEM containing 10% FBS (Invitrogen) to serum-freemedium by direct transfer. Briefly, sub-confluent, logarithmic cellswere trypsinized, washed once with serum-free medium and inoculateddirectly into 250 ml Erlenmeyer flasks with a 0.2μ filter (Corning),containing 25 ml of ExCell-525 serum-free medium (JRH Biosciences) at astarting cell concentration of 0.3-0.5×10₆ ml⁻¹, unless otherwise noted.Cultures were maintained in logarithmic growth in Erlenmeyer flasks bypassage every two to three days. Flasks were shaken on a magnetic shakerplatform (Infors) at 100 rpm in a humidified incubator at 37° C. and 5%CO₂. Cultures were passaged by centrifugation at 1000 rpm for 5 minutes.The supernatant was removed and the pellet re-suspended in the remainingmedium. Fresh, cold medium (4° C.) was added and new flasks inoculatedat the appropriate cell concentration. After transfer to serum-freemedium, cultures were passaged for two to four weeks to allow forcomplete adaptation, after which a serum-free cell bank was created. Allexperiments were started using cells from this cell bank.

Bioreactors

Bioreactor cultures were performed in 3 L reactors with a 2 L workingvolume (Applikon). Temperature was maintained at 37° C. by a heatingblanket. Dissolved oxygen concentration (dO₂) was controlled at 50% ofair saturation by adjusting inlet gas composition through the headspaceand intermittent sparging through a microporous sparger. Startingculture pH was controlled at 7.3 by CO₂ addition through the microporoussparger. The lower culture pH limit was set at 6.7 so that the culturepH was allowed to drift downwards (the lower limit was not reached).Cultures were agitated by two marine impellers at 75 rpm. Process datawas acquired by the BioExpert software (Applikon).

Analytical Protocols

Cell counts and viability measurements were performed using a CASYautomatic cell counter (Schärfe Systems). Glucose, lactate, ammonia andphosphate concentrations were determined using an EKTACHEM II® analyzer(Kodak) with cell-free culture supernatants. Amino acid concentrationswere determined using a modified AccuTag HPLC method (Waters) asdescribed by van Wandelen and Cohen (1997). Aliquots (200 μl) ofcentrifuged culture supernatant were stored at −20° C. in 1 ml cryovials(Nalgene) until required. Samples from each experiment were analyzed atthe same time to avoid experimental variation. Osmolality was measuredby a freezing point depression osmometer (Osmomat 030-d, Gonotec).Antibody concentration was determined by a sandwich-type ELISA. Briefly,plates were coated with 2 μg ml⁻¹ mouse anti-human IgG against the kappalight chain (Pharmingen) and incubated overnight at 4° C. AnHRP-conjugated mouse anti-human IgG against the heavy chain (Pharmingen;1:500) was used as detection antibody for one hour at 37° C. with OPD(Sigma) as substrate. Washing between incubation steps was performedwith 0.05% Tween 20 in PBS. Samples were diluted in washing buffersupplemented with 0.1% BSA. Quantification was relative to an IgG1reference standard using a calibration range of 10 to 400 ng ml⁻¹.Antibody samples purified by Protein A were subject to quality analysisby isoelectric focusing (IEF) and denaturing polyacrylamide gelelectrophoresis (SDS-PAGE). For glycan analysis, N-linked glycans wereremoved by PNGase F treatment of the IgG samples in 20 mM sodiumphosphate (pH 7.2) and analyzed with MALDI-MS in the reflector mode onan Applied Biosystems Voyager DE Pro mass spectrometer. The matrix was2,5-dihydroxybenzoic acid (10 mg ml⁻¹) in 50/50/0.1acetonitrile/water/trifluoroacetic acid. Spectra were obtained in thepositive ion mode and glycans were detected as sodium adducts, [M+Na]+.

Calculation of Cell Specific Metabolic Rates

Cell specific rates of metabolite utilization and production in batchand fed-batch culture were calculated using the log mean of the cellconcentration as shown in the following equation:q _(S)=(C ₂ −C ₁)/(t ₂ −t ₁)×[(X ₂ −X ₁)/1n(X ₂ −X ₁)].

In this equation, C is the metabolite concentration (μmoles/l), t istime (days) and X is the viable cell concentration. A rate constantaccounting for the spontaneous decomposition of glutamine was notincluded as decomposition was not significant at the time points atwhich the rates were calculated (data not shown). The yield coefficientsof lactate produced per glucose (Y_(lac/glc)), ammonia produced perglutamine (Y_(amm/gln)) and alanine produced per glutamine (Y_(ala/gln))were calculated from the equations below and are expressed in mole/mole:Y _(lac/glc) =q _(lac) /q _(glc)Y _(amm/gln) =q _(amm) /q _(gln)Y _(ala/gln) =q _(ala) /q _(gln)

EXAMPLES Example 1 Increasing Maximum Final Cell Yields in Batch Cultureof PER.C6 Cells

The simplest production process is a batch culture. However, this isrestricted in the viable cell concentration and therefore the productyields attainable, due largely to nutrient limitation. A method ispresented to increase the maximum final cell concentration of a batchculture of PER.C6® or PER.C6-derived sub-clones by calculating the cellspecific rate of utilization of key nutrients at different cellconcentrations and starting the batch culture at a cell concentrationwhere there is optimal utilization of nutrients with respect to cellgrowth.

The DNA encoding the antigen-binding region of an antibody recognizingepithelial cell adhesion molecule (EpCAM) was first isolated from a scFvphage display library (Huls et al., 1999). DNA encoding theantigen-binding region of an antibody recognizing CD46 was isolated asdisclosed in WO 02/018948, the contents of which are incorporated bythis reference. A leader sequence and constant regions of IgG1 type wereadded essentially as described in Boel et al., 2000. The DNA encodingthe light and heavy chains were then cloned into expression vectorpcDNA3002(Neo). The expression vector pcDNA3002(Neo), which has beendescribed in international patent application PCT/NL02/00841, wasdeposited on Dec. 13, 2001, at the European Collection of Cell Cultures(ECACC) under Number 01121318. The resulting expression vectors,encoding an IgG1 that recognizes EpCAM or CD46, respectively, regulatedby a CMV promoter, was introduced in PER.C6 cells according to standardmethods.

A recombinant antibody-expressing clone, derived from a parentalpopulation of the PER.C6® cell line, was used in these experiments. Theclone expressing anti-EpCAM is further referred to herein as clone 1,the clone expressing anti-CD46 is further referred to herein as clone 2.

Cells were maintained in EXCELL™ 525 medium (JRH Biosciences)(maintenance of the cells in GTM-3 medium (Sigma) did also work) andbatch productions were carried out in EXCELL™ VPRO medium (JRHBiosciences, Cat. No. 14560). Cells were transferred directly fromEXCELL™ 525 to EXCELL™ VPRO for the batch productions.

FIG. 1 shows that the maximum final viable cell concentration ofcultures started at 1×10⁶ cells ml⁻¹ reached almost 14×10⁶ cells ml⁻¹after six days (approximately three-fold higher than batch cultures ofCHO and Sp2/0), compared to cultures started at 0.3×10⁶ ml⁻¹, whichreached 10×10⁶ cells ml⁻¹ after nine days. There is very littledifference in the final antibody titers of both cultures. However, inthe culture started at 1×10⁶ ml⁻¹, approximately 600 mg L⁻¹ was reachedafter six days, compared to nine days for the cultures started at0.3×10⁶ ml⁻¹.

The higher cell concentrations observed in cultures started at 1×10⁶cells ml⁻¹ compared to 0.3×10⁶ ml⁻¹ is due to the lower cell specificrate of nutrient utilization at the higher cell concentration. Therespiration rate of hybridoma cells has been shown to decrease withincreasing cell density (Wohlpart et al., 1990). Similarly, the cellspecific rate of utilization of a nutrient has also been shown todecrease with increasing cell concentration (Portner et al., 1994,Yallop and Svendsen 2001). We have now used this information in a noveland inventive way to form a concept for increasing attainable celldensities in a culture.

By calculating the cell specific rate of utilization of a key nutrienteach day in a batch culture and plotting these values against cellconcentration, a graph can be obtained as shown in FIG. 2 for glutamine.FIG. 2 shows the relationship between the cell specific rate ofglutamine utilization (q_(Gln)) and cell concentration. From this graph,an optimum starting cell concentration can be selected based on optimaluse of the available nutrients. For example, a culture starting at0.3×10⁶ cells ml⁻¹ will reach approximately 0.5×10⁶ ml⁻¹ in 24 hours(average population doubling time (pdt) of this clone is 32 hours). Theq_(Gln) value at 0.5×10⁶ cells ml⁻¹ is approximately 2.5 μmoles 10⁶cells⁻¹ 24 hours⁻¹. The total glutamine consumed in this 24 hours will,therefore, be approximately 1.25 μmoles ml⁻¹ (0.5×2.5). However, aculture starting at 1×10⁶ cells ml⁻¹ will reach approximately 1.5×10⁶ml⁻¹ in 24 hours. The q_(Gln) value at this cell concentration isapproximately 0.75 μmoles 10⁶ cells⁻¹ 24 hours⁻¹. The total glutamineconsumed will therefore be approximately 1.125 μmoles ml⁻¹. The twocultures will therefore use approximately the same amount of glutaminein the first 24 hours.

It is, therefore, another object of the invention to provide a method ofculturing cells, comprising starting a culture at a cell concentrationwhere the specific nutrient utilization level is close to a minimumplateau level. This equates with around 0.8 to 2.0×10⁶ cells/ml,preferably, 0.9-1.5×10⁶ cells/ml, for E1-immortalized retina cells,particularly, PER.C6-derived cells. It is, therefore, an embodiment ofthe invention to subculture the cells at a seeding concentration of0.8-2.0×10⁶ cells/ml, preferably, 0.9-1.5×10⁶ cells/ml, more preferably,0.95-1.25×10⁶ cells/ml.

The advantage of this aspect of the invention is that the number ofviable cells that can be obtained is higher at this higher seedingdensity, and higher numbers of cells are reached faster during theprocess. This aspect of the invention, therefore, is very useful forbatch cultures, but can also be beneficially used in fed-batch culturesor (fed-) perfusion cultures, such as those of the present invention.

Example 2 Feed Strategies for Improving Antibody Yields in PER.C6Derived Sub-Clones

Fed-batch processes aim at increasing product yields by increasing theviable cell concentration or prolonging the production period by feedingnutrient concentrates to replenish those that are consumed. We presenthere a feed strategy for improving the antibody yields of PER.C6®derived sub-clones. The feed strategy can be combined with a higherstarting cell density to obtain a higher final cell density at the onsetof the nutrient feed and a shorter overall production process.

A basic nutrient feed concentrate consisting of glucose, phosphate,glutamine and the 15 other amino acids was prepared based on thenutrient utilization profile of six duplicate batch cultures of clone 1in shake-flask (see, e.g., FIG. 3). Similar utilization profiles wereobserved for clone 2, and, hence, it is expected that the feed strategydescribed below for clone 1 will also improve yields from other clones,thereby providing a more generic strategy for fed-batch or fed-perfusioncultures of E1-immortalized cells, preferably, retina cells, preferably,cells derived from PER.C6 cells. The concentrate is listed in Table 1.Optionally, calcium and three recombinant growth factors, LongR3 GF-1,Long EGF and insulin were also added to the feed. At this point, theaddition of calcium and the growth factors did not significantlyinfluence the results that were obtained. Glycine appeared not essentialfor the feed, and was no longer added in later experiments. Insulin waspurchased from Sigma, LongR3 IGF-1 and Long EGF were purchased fromGroPep. All amino acids were purchased from Sigma. The timing andfrequency of addition of the feed concentrates was varied. The time ofthe first addition was tested at 0, 1, and 2 days prior to nutrientexhaustion. Glucose and phosphate were used as indicators for the startof the feed. A series of bolus additions were made every two days, basedon the predicted viable cell concentration. Usually, six feeds wereprovided. The concentrations of the added components as presented inTable 1 do not take into account the remaining component in the spentmedium before the addition (i.e., the concentration of a component afteraddition into the culture medium will be higher than that provided inTable 1, because before the addition the culture medium will stillcontain some of this component, as additions, according to theinvention, are done before the component is completely used up by thecells).

FIG. 4 shows the effect of feeding the concentrate mix to a sub-clone ofPER.C6® expressing a recombinant antibody (clone 1). Starting the feedat day 3 (two days prior to nutrient exhaustion and continuing every twodays after this) resulted in a final antibody yield of approximately 800mg L⁻¹, an increase of approximately 1.6-fold over the batch process,which gave 500 mg L⁻¹. Starting the feed at day 5 and continuing everytwo days after this) resulted in a similar increase in final antibodyconcentration.

Osmolality in the batch cultures (Example 1) decreased from 280 to 240mOsm Kg⁻¹, while in the feed cultures, it increased, eventually risingto 300-310 mOsm Kg⁻¹.

Example 3 Achieving viable cell numbers above 30×10⁶ cells per ml andantibody yields above 500 mg L⁻¹ day⁻¹

Fed-batch processes may result in a build-up of toxic metabolites suchas lactate and ammonia and an increase in medium osmolarity, whicheventually limit the viable cell concentration and the length of theprocess, thus, impacting on product yields. A possible alternative to afed-batch process is a perfusion process, where high cell concentrationscan be maintained by a continual medium exchange and a cell bleed(removing a certain percentage of the cells population). A possibledrawback with such a process is a relatively low product concentrationdue to the large volumes of medium that are required, the relatively lowcell viability often encountered and the relatively high level ofcomplexity to operate such a system. It is, therefore, only advantageousto operate perfusion processes if very high viable cell concentrationsand/or specific productivities can be maintained.

We present here the attainment of a viable cell concentration above30×10⁶ ml⁻¹ and antibody yields of above 600 mg L⁻¹ 24 hours⁻¹ in shakeflask cultures with one medium volume exchange per day.

Logarithmic cultures of antibody producing PER.C6® cells, cultured inshake flask with EXCELL™ 525 were transferred into shake flaskscontaining EXCELL™ VPRO at a starting cell number 1×10⁶ cells/ml (otherstarting cell concentrations gave similar results). Medium replacementby centrifugation (one volume per day) was started at day 3-5. No cellbleed was operated. Samples for metabolite analysis, antibodyquantification, and cell counts were taken every day and stored at −20°C.

FIG. 5 shows that a viable cell number of up to 50×10⁶ ml⁻¹ and anantibody yield of 500-750 mg L⁻¹ 24 hours⁻¹ was maintained for at leastfive days without a cell bleed, for two independent antibody producingcell clones. Viability of the cells was around 80-90%. These high celldensities are approximately three-fold higher than is generallyachievable with other cell lines like CHO and Sp2/0, and, hence, retinacells that are immortalized with adenovirus E1 sequences, such asPER.C6® cells, are very suitable for perfusion processes. A cell bleedwill improve the length of the process, and, therefore, an optimizedsystem may include one or more cell bleed steps.

Up to 50×10⁶ cells per ml, with a viability of around 80-90%, could bemaintained for at least five days with one complete medium change everytwo days. With this strategy, many of the nutrients became depleted onthe second day. The medium is therefore, preferably, changed daily. In aperfusion process, this could translate into a change of about 1 to 3volumes/day. This is near the typical range in a standard perfusionsystem, where the medium is changed at about 0.5 to 2 volumes/day. Thesomewhat higher values for the cells, according to the invention, aredue to the very high cell concentrations with the cells of the inventionin a perfusion system. When cell concentrations of more than 30×10⁶cells/ml according to the invention are preferred, the medium exchangeshould at least be 0.5 culture volumes/day, preferably, at least oneculture volume/day. Failure to supply the nutrients (here via theculture medium) in sufficient concentration leads to cell death. Thedaily medium change results in higher viable cell densities (up to50×10⁶ cells/ml with daily medium change vs. 10×10⁶ cells/ml withoutdaily medium change, see FIGS. 1 and 4). Furthermore, with a dailymedium exchange, the cells give similar product yields in one day asachieved in a batch process of 8 to 13 days.

Example 4 Feed Strategies for Further Improving Antibody Yields inPER.C6 Derived Sub-Clones

The provision of a balanced nutrient feed extends to components such asvitamins, trace elements and lipids. Concentrates (10× or 50×, bothworked) of EXCELL® VPRO vitamins, inorganic salts, trace elements,growth factors, lipids and plant hydrolysates were obtained from JRHBiosciences and added together with the basic feed concentrate (minuscalcium and growth factors) described in Example 2. The EXCELL® VPROconcentrates were added to give a final concentration of 0.25×.

FIG. 6 shows the results of this modified feed on the growth (FIG. 6A)and antibody yields (FIG. 6B) of clone 1 in shake-flask versus a batchcontrol. The results were obtained by starting the feed at day 3 (48hours prior to nutrient depletion). Starting the feed at day 5 (day ofnutrient depletion) gave similar results. The viable cell number wasmaintained for significantly longer than the batch control and antibodyyields increased 2.0-fold from 0.5 g L⁻¹ in the batch to 1.0 g L⁻¹ inthe fed-batch process.

Spent medium analysis of these feed experiments identified a change inthe cell specific rates of utilization of some of the amino acids, whichappeared to be due to the addition of the VPRO concentrates. The aminoacid concentrate listed in Example 2 was, therefore, modified as shownin Table 2. The feed was started 48 hours prior to nutrient depletionand additions were made every two days. Usually, six feeds wereprovided. Again, the concentrations of the added components as presentedin Table 2 do not take into account the remaining component in the spentmedium before the addition.

For the first feed addition, increased concentrations of glutamine andserine were used as compared to the subsequent feeds (see Table 2).Phosphate and glucose were used as markers to determine the start of thefeed. Clones 1 and 2 were used in this experiment.

Experiments were carried out in shake-flask and bioreactor. Shake flaskexperiments were carried out as described. Bioreactor experiments wereinitiated by inoculating a 3 L bioreactor (Applikon, 2 L working volume)with cells from a logarithmic pre-culture grown in shake flask. Thepre-culture and bioreactor experiments were performed in EXCELL® VPRO.The split ratio for inoculation into the bioreactor was at least 1:6,and the seeding cell concentration was about 0.3×10⁶ cells/ml.

Results

FIG. 7 shows the results of the modified feed on clone 1 in bioreactorversus a batch control. The maximum viable cell number reached 10-12×10⁶ml⁻¹ and viable cell numbers were maintained between 8 and 10×10⁶ cellsml⁻¹ until the end of the culture at day 19 (FIG. 7A). Antibody yieldsincreased three-fold from 0.4 g L⁻¹ in the batch to 1.3 g L⁻¹ in thefed-batch process (FIG. 7B).

Osmolality and ammonia reached 430 mOsm Kg⁻¹ and 16 mmoles L⁻¹,respectively, in these feed cultures, levels that have been reported ashaving negative effects on culture performance and product quality. Itmay, therefore, be that the decrease in viable cell numbers observedtowards the end of the process was due, at least in part, to thesefactors.

FIG. 8 shows the results of the feed strategy on clone 2 in 2 Lbioreactors. Maximum viable cell numbers reached 10-11×10⁶ ml⁻¹ and7-9×10⁶ ml⁻¹ were maintained until the end of the culture at 19 days.Antibody yields were increased three-fold from 0.5 g L-1 to 1.5 g L⁻¹.

A third clone expressing another, again unrelated, antibody wassubjected to the same batch process and the fed-batch process with thesame feed strategy. FIG. 10 shows the results of the feed strategy onthis clone 3 in shake flask. Maximum viable cell numbers reached 14×10⁶ml⁻¹ and 10-12×10⁶ ml⁻¹ were maintained until the end of the culture onday 17. Antibody yields were increased three-fold from 0.7 g L⁻¹ to 2.1g L⁻¹.

The feed strategy, therefore, improves the yield for different clonesthat each express a different antibody, indicating that the process,according to the invention, is generically applicable.

It is, therefore, an aspect of the invention to provide a processcomprising the feed strategy, according to the invention, wherein theyield of a produced protein is increased at least 1.5×, preferably, atleast 2×, more preferably, at least 2.5×, still more preferably, atleast 3× over the yield in the batch process.

The specific productivity (q_(Ab)) of the cells used in the presentinvention was approximately between 12 to 18 pg antibody/cell/day. Insome instances the q_(Ab) was around 10 pg antibody/cell/day, and inother instances values up to about 25 pg antibody/cell/day were observedwith the cells and methods of the present invention. In the batchcultures, this decreased significantly before maximum cell numbers werereached, coinciding with depletion of nutrients, which was approximatelyafter seven days, whereas in fed-batch cultures this specificproductivity was kept at this level until two to three days after thelast feed addition, which amounted to around 16 to 18 days, according toa process of the invention.

Product Quality

In the experiments described above, product quality was checked byvarious methods, including iso-electric focusing, SDS-PAGE, MALDI-TOFmass spectrometry and HPAEC-PAD. In all cases, the produced antibodybasically showed a human-type glycosylation and the structural integrityof the produced antibodies was very good, irrespective of the processused, and very similar to that reported in (Jones et al., 2003), whereboth cell numbers and product yields were lower. Therefore, theincreased yields obtainable by processes of the invention were notobtained at the cost of a significant decrease in product quality.

Protein A purified IgG produced from batch and fed-batch cultures wasanalyzed by MALDI-MS. Material produced by PER.C6 cells from batchcultures showed a galactosylation profile similar to that shown by IgGpurified from human serum and no hybrid or high mannose structures wereidentified in either batch or fed-batch produced material. The averagepercentage of glycans terminating in 0, 1, and 2 galactose residues(G0:G1:G2) from all the batch cultures tested was 29, 54, and 17%,respectively. This can be compared to CHO and hybridoma producedantibody, which is often predominantly in the G0 form. For example,Hills et al. (1999) reported a galactosylation profile (G0:G1:G2) for anantibody produced in NS0 and CHO cells.

Antibody produced in the fed-batch process showed a reduced level ofgalactosylation compared to the batch (FIG. 9). The percentage of G0glycoforms increased from 29 to 49%, while the G1 and G2 glycoformsdecreased from 54% and 17% to 42% and 9%, respectively. This decrease ingalactosylation was probably due to the high (up to 16 mM) ammoniaconcentrations at the end of the fed-batch cultures. However, the levelof galactosylation in the antibody produced by the fed-batch process inPER.C6 cells was still higher than typically seen in batch-producedantibodies from CHO, for example, (Hills et al., 1999). Isoelectricfocusing (IEF) and SDS-PAGE revealed no significant differences betweenthe material produced by batch or fed-batch cultures (data not shown)and in all cases, aggregation was below 3%.

Despite relatively low Y_(amm/gln) values, the high viable cellconcentrations resulted in a supply of glutamine in the feed such thatthe ammonia accumulated up to 16 mmoles L⁻¹. While this did not resultin a drop in the viable cell concentration, batch cultures initiated inthe presence of NH₄Cl showed that concentrations above 9 mmole L⁻¹negatively affected growth rates and maximum cell concentrations.Furthermore, glycosylation was also somewhat affected (see FIG. 9). Itmay, therefore, be beneficial to reduce ammonia accumulation, e.g.,according to a method described below.

Two areas for attention in the process described so far are the highlevels of ammonia and osmolality. A large contributor to the increase inosmolality came from the VPRO (medium) concentrates. An approach toreduce this osmolality is, therefore, to identify which of the mediumcomponent groups (vitamins, trace elements, inorganic salts, growthfactors etc.) are important to culture performance and remove those thatare not important. This should benefit the process, not only by reducingthe osmolality of the feed, but also by removing any potentiallydeleterious components and by allowing the optimization of addition ofthe most important components. It would also reduce the cost of thefeed.

Reduction in ammonia accumulation may be achieved by more strictlycontrolling glutamine addition. This can be done based on thecalculations of the specific consumption and cell numbers, as describedsupra. This can be achieved by continuously pumping in glutamine at anappropriate rate, matched to the viable cell concentration and the cellspecific rate of utilization, so that residual glutamine concentrationsin the medium are maintained at a constant low level, such as between0.2 and 1.5 mM, preferably, between 0.5 and 1.0 mM. Another approachthat may be possible for the cells, according to the invention, is theremoval of glutamine from the feed when the ammonia concentrationreaches a certain point, e.g., in one or more of the feeds, subsequentto the first feed, so that the cells are forced to switch to glutaminesynthesis using ammonia and glutamate and the glutamine synthetasepathway. This approach is not generally possible for cell types such asBHK and CHO, as glutamine depletion often results in rapid andwidespread cell death and transfer to glutamine-free conditions oftenrequires a period of adaptation. However, in batch cultures of thecells, according to the present invention, the viable cell concentrationcontinued to increase for two days after the depletion of glutamine andculture viability was not significantly affected, suggesting that theremay be sufficient flux through the glutamine synthetase pathway at leastto maintain the culture.

Spent medium analysis of the most optimized fed-batch culture (examplesin FIGS. 7, 8) showed that only cystine was depleted during the process.A further modification of the amino acid feed, according to theinvention, is, therefore, an increase in the cystine concentration,e.g., to 0.3 to 0.35 mmoles/l or even up to 0.6 mmoles/l for every10×10⁶ cells/ml.

Example 5 Improved (Fed-)Batch Process

Feed concentrates developed for fed-batch processes may also be used tosupplement culture media for use in an improved batch process.Supplementing a culture medium with at least one of the feed additionsfrom a fed-batch process has been shown by others to improve batchyields. A similar approach of supplementing culture media with feedconcentrates may also be used to reduce the number of feed additionsduring a fed-batch process, thereby simplifying the process, as alsoshown by others.

The present invention discloses feed strategies for cells that have beenimmortalized by adenovirus E1 sequences, such as PER.C6 cells. It isshown herein which components become limiting in a fed-batch process,and the amounts of, as well as the ratio between, the components thatcan be added to improve yields in a fed-batch process are disclosedherein. This information is used, in this example, to provide animproved batch process. It is assumed that such a culture will containabout 10×10⁶ cells/ml, as this is around the number of cells that hasbeen observed in the batch and fed-batch cultures of the invention. Inthe fed-batch experiments, six feeds were added, with concentrations ofthe components as in Tables 1 or 2. The addition of 10% to 60% of thetotal (i.e., the total of all six feeds together) feed, preferably, 20%to 40% of the total feed, results in an improved batch process, becausethe nutrients will become depleted later during the culture, and, hence,the yields will go up because of prolonged productivity compared to thestraight batch process disclosed above, where no additions are made tothe culture medium. The components can be added directly to the culturemedium at any stage prior to depletion of nutrients from the medium, butare, preferably, added prior to start of the culture so that no otheradditions have to be made during the process (improved batch process),which makes the process very simple. Of course, this may be combinedwith extra additions of certain components later during the process(fed-batch process), in which case less additions have to be made tomake the process than in the fed-batch process disclosed above, therebyproviding a simpler fed-batch process. It is, therefore, anotherembodiment of the invention to provide a method for producing a productin cells immortalized by adenovirus E1 sequences, wherein the cells arecultured in a culture medium, characterized in that the followingcomponents are added to the culture medium in the following amounts:glucose (3.6-21.6 mmoles/l, preferably, 7.2-14.4 mmoles/l), glutamine(6.8-40.9 mmoles/l, preferably, 13.6-27.2 mmoles/l), leucine (0.40-2.4mmoles/l, preferably, 0.79-1.6 mmoles/l), serine (2.31-13.9 mmoles/l,preferably, 4.62-9.24 mmoles/l), isoleucine (0.3-1.8 mmoles/l,preferably, 0.6-1.2 mmoles/l), arginine (0.28-1.66 mmoles/l, preferably,0.55-1.10 mmoles/l), methionine (0.14-0.83 mmoles/l, preferably,0.28-0.55 mmoles/l), cystine (0.15-0.9 mmoles/l, preferably, 0.3-0.6mmoles/l), valine (0.27-1.62 mmoles/l, preferably, 0.54-1.08 mmoles/l),lysine (0.26-1.58 mmoles/l, preferably, 0.53-1.06 mmoles/l), threonine(0.18-1.08 mmoles/l, preferably, 0.36-0.72 mmoles/l), asparagine(0.06-0.36 mmoles/l, preferably, 0.12-0.24 mmoles/l), tyrosine(0.078-0.47 mmoles/l, preferably, 0.16-0.31 mmoles/l), histidine(0.06-0.36 mmoles/l, preferably, 0.12-0.24 mmoles/l), phenylalanine(0.012-0.072 mmoles/l, preferably, 0.024-0.048 mmoles/l), tryptophan(0.036-0.22 mmoles/l, preferably, 0.072-0.14 mmoles/l) and phosphate(0.45-2.7 mmoles/l, preferably, 0.9-1.8 mmoles/l). The amounts betweenbrackets are 10% to 60%, preferably, 20% to 40%, of the amounts of 6×the feeds of Table 2. Preferably, also culture medium concentrate (10×,50×, or other suitable concentrates can be used) is added to an endconcentration of between 0.15×-0.9×, preferably, between 0.3×-0.6×.Preferably, the culture medium in these embodiments is ExCell VPROmedium. An amount of 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 single feeds (asingle feed being an amount as disclosed in Table 1 or 2) is added toculture medium, and simple batch processes for culturing the cells ataround 10×10⁶ cells/ml and producing product (e.g., antibody), accordingto the invention, are performed with the, thus, fortified media, todetermine the optimum amount of component additions. Improved batchprocesses giving the highest product yields are expected when about 20%to 40% of the total feed of a fed-batch process, according to theinvention, are provided to the culture medium prior to culturingsomewhere between 1 to 2.5 single feeds. Of course, more fine-tuning ofthe amount is possible once a beneficial range of added components isestablished by these experiments. Of course, when the cell numbers aredifferent, the component addition can again be adapted. For instance, ifthe cells are cultured at a density of only 5×10⁶ cells/ml, addition ofan amount of only half the amount above would be required, as is clearto the person skilled in the art. TABLE 1 Final Concentration (afteraddition) Components (per 10 × 10⁶ cells/ml) (mmoles L⁻¹) Glucose 6.00Glutamine 1.75 Leucine 0.60 Serine 0.55 Isoleucine 0.45 Arginine 0.42Methionine 0.23 Cystine 0.14 Valine 0.45 Lysine 0.40 Threonine 0.33Glycine 0.33 Asparagine 0.15 Tyrosine 0.14 Histidine 0.11 Penylalanine0.10 Tryptophan 0.02 Phosphate 0.70 Calcium 0.02* LongR3 IGF-1 50 ug/L*Long EGF 50 ug/L* Insulin 20 ug/L**optionally present

TABLE 2 Final Concentration (after addition) (per 10 × 10⁶ cells/ml)(mmoles L⁻¹) Components First Feed Subsequent Feeds Glucose 6.00 6.00Glutamine 2.60 1.75 Leucine 0.66 0.66 Serine 1.10 0.55 Isoleucine 0.500.50 Arginine 0.46 0.46 Methionine 0.23 0.23 Cystine 0.25 0.23 Valine0.45 0.45 Lysine 0.44 0.44 Threonine 0.30 0.30 Asparagine 0.10 0.10Tyrosine 0.13 0.13 Histidine 0.10 0.10 Penylalanine 0.02 0.02 Tryptophan0.06 0.06 Phosphate 0.75 0.75 10X VPRO Concentrate 0.25X 0.25X

REFERENCES

-   Boel E., S. Verlaan, M. J. Poppelier, N. A. Westerdaal, J. A. Van    Strijp, and T. Logtenberg (2000). Functional human monoclonal    antibodies of all isotypes constructed from phage display    library-derived single-chain Fv antibody fragments. J. Immunol.    Methods 239:153-66.-   Hills A. E., A. K. Patel, P. N. Boyd and D. C. James (1999). Control    of therapeutic antibody glycosylation. In: A. Bernard, B.    Griffiths, W. Noe and F. Wurm (eds), Animal Cell Technology:    Products from Cells, Cells as Products, 255-257. Kluwer Academic    Press, Dordrecht, The Netherlands.-   Huls G. A., I. A. F. M. Heijnen, M. E. Cuomo, J. C.    Koningsberger, L. Wiegman, E. Boel, A-R van der Vuurst-de    Vries, S. A. J. Loyson, W. Helfrich, G. P. van Berge Henegouwen, M.    van Meijer, J. de Kruif, and T. Logtenberg (1999). A recombinant,    fully human monoclonal antibody with antitumor activity constructed    from phage-displayed antibody fragments. Nat. Biotechnol.    17:276-281.-   Jones D., N. Kroos, R. Anema, B. Van Montfort, A. Vooys, S. Van Der    Kraats, E. Van Der Helm, S. Smits, J. Schouten, K. Brouwer, F.    Lagerwerf, P. Van Berkel, D-J Opstelten, T. Logtenberg, and A. Bout    (2003). High-level expression of recombinant IgG in the human cell    line PER.C6. Biotechnol. Prog. 19:163-168.-   Portner R., A. Bohmann, I. Ludemann and H. Markl (1994). Estimation    of specific glucose uptake rates in cultures of hybridoma cells. J.    Biotechnol. 34:237-246.-   Sauer P. W., J. E. Burky, M. C. Wesson, H. D. Sternard and L. Qu    (2000). A high yielding, generic process fed batch cell culture    process for production of recombinant antibodies. Biotechnol.    Bioeng. 67:585-597.-   Tanase T., Y. Ikeda, K. Iwama, A. Hashimoto, T. Kataoka, Y.    Tokushima and T. Kobayashi (1997). Comparison of micro-filtration    hollow fiber bioreactors for mammalian cell culture. J. Ferm. Ioeng.    83:499-501.-   Xie L., W. Pilbrough, C. Metallo, T. Zhong, L. Pikus, J. Leung,    Aunin{hacek over (s)} and W. Zhou (2002). Serum-free suspension    cultivation of PER.C6® cells and recombinant adenovirus production    under different pH conditions. Biotechnol. and Bioengin. 80:569-579.-   Weidemann R., A. Ludwig and G. Kretzmer (1994). Low temperature    cultivation—A step towards process optimization. Cytotechnology    15:111-116.-   Wohlpart D., D. Kirwan and J. Gainer (1990). Effects of cell density    and glucose and glutamine levels on the respiration rates of    hybridoma cells. Biotechnol. Bioeng. 36:630-635.-   Yallop C. A. and I. Svendsen (2001). The effects of G418 on the    growth and metabolism of recombinant mammalian cell lines.    Cytotechnology 35:101-114.

1.-20. (canceled)
 21. A culture of human embryonic retina (HER) cellsimmortalized by adenovirus E1 sequences, wherein said culture of cellscomprises at least 10×10⁶ cells/ml.
 22. The culture of cells of claim21, wherein said culture of cells comprises at least 12×10⁶ cells/ml.23. The culture of cells of claim 22, wherein said culture of cellscomprises at least 15×10⁶ cells/ml.
 24. The culture of cells of claim23, wherein said culture of cells comprises at least 20×10⁶ cells/ml.25. The culture of cells of claim 24, wherein said culture of cellscomprises at least 30×10⁶ cells/ml.
 26. The culture of cells of claim25, wherein said culture of cells comprises at least 40×10⁶ cells/ml.27. The culture of cells of claim 21, wherein at least 80% of the cellsare viable.
 28. A culture of cells according to claim 21, whereinculture medium is exchanged at a rate of about 0.2-3 culturevolumes/day.
 29. The culture of cells of claim 21, wherein cells in saidculture of cells express a recombinant protein.
 30. The culture of cellsof claim 29, wherein said cells produce at least 10 pg protein/cell/day.31. The culture of cells of claim 21, wherein said culture of cells is abatch culture and wherein cells in said culture of cells express arecombinant immunoglobulin with a yield of at least 500 mg/l.
 32. Theculture of cells of claim 30, wherein said yield is at least 700 mg/l.33. The culture of cells of claim 29, wherein said culture of cells is aperfusion or fed-perfusion culture and wherein said recombinant proteinis an immunoglobulin that is expressed with a yield of at least 150mg/l/day.
 34. The culture of cells of claim 21, wherein said cells arePER.C6 cells as represented by the cells deposited under ECACC No.96022940.
 35. The culture of cells of claim 21, wherein said culture ofcells is a suspension culture. 36.-41. (canceled)